Ultrasmall cerium oxide nanoparticles as highly sensitive X-ray contrast agents and their antioxidant effect

Owing to their theranostic properties, cerium oxide (CeO2) nanoparticles have attracted considerable attention for their key applications in nanomedicine. In this study, ultrasmall CeO2 nanoparticles (particle diameter = 1–3 nm) as X-ray contrast agents with an antioxidant effect were investigated for the first time. The nanoparticles were coated with hydrophilic and biocompatible poly(acrylic acid) (PAA) and poly(acrylic acid-co-maleic acid) (PAAMA) to ensure satisfactory colloidal stability in aqueous media and low cellular toxicity. The synthesized nanoparticles were characterized using high-resolution transmission electron microscopy, X-ray diffraction, Fourier transform-infrared spectroscopy, thermogravimetric analysis, dynamic light scattering, cell viability assay, photoluminescence spectroscopy, and X-ray computed tomography (CT). Their potential as X-ray contrast agents was demonstrated by measuring phantom images and in vivo CT images in mice injected intravenously and intraperitoneally. The X-ray attenuation of these nanoparticles was greater than that of the commercial X-ray contrast agent Ultravist and those of larger CeO2 nanoparticles reported previously. In addition, they exhibited an antioxidant effect for the removal of hydrogen peroxide. The results confirmed that the PAA- and PAAMA-coated ultrasmall CeO2 nanoparticles demonstrate potential as highly sensitive radioprotective or theranostic X-ray contrast agents.

5][16][17] The natural radiation dose is 2-3 mSv per year. 18Each medical CT scan covers 0.001-16 mSv, depending on the scanning objects of the body; hence, multiple CT scans are harmful to the body. 19ontrast agents can reduce the X-ray radiation dose without deteriorating the image quality via contrast enhancement. 12,13hey also facilitate the identication and diagnosis of certain conditions and diseases of the body. 12,13Currently, the iodine contrast agents approved by the United States Food & Drug Administration 6,20 exhibit limitations, such as low sensitivity, necessitating high injection doses that could cause side effects, 21 and low contrast for so tissues.In addition, they undergo rapid renal excretion because of their low molecular masses, allowing only brief imaging times.However, heavy metal-based nanoparticles can overcome these limitations because of their higher X-ray attenuation, 22 lower osmolality and viscosity, 6,23 and longer blood vessel circulation times 24 than those of molecular iodine contrast agents, leading to higher contrast images, lower injection doses, and longer imaging times.Therefore, developing alternative contrast agents derived from heavy metal-based nanoparticles is imperative.
][31] Thus far, a limited number of Ce-containing nanoparticles have been reported as radioprotective 15 or theranostic [32][33][34][35][36] X-ray contrast agents.Based on the high X-ray attenuation of CeO 2 nanoparticles 22 and their exceptional catalytic properties, rendering them highly effective in removing excess ROS from radiation-induced damage, [26][27][28] Garcia et al. synthesized 5 nm albumin-stabilized CeO 2 nanoparticles and used them for the in vivo imaging of normal and tumor-model mice. 15Chaurand et al. successfully located CeO 2 nanomaterials [particle diameter (d) = ∼31 nm] in mouse lung tissue using X-ray imaging. 32hey reported that the X-ray attenuation was ∼2 times greater than that of the commercial iodine contrast agent Iohexol.Liu et al. synthesized CeO x nanoparticles embedded in mesoporous silica particles (overall diameter = 119-134 nm) and applied them for the diagnosis and X-ray induced photodynamic therapy of cancer. 33They reported that the X-ray attenuation was 3.79 times greater than that of the iodine contrast agent Iohexol.Cao et al. synthesized dextran-coated CeO 2 nanoparticles (d = 3 nm) and applied them to CT-guided therapy of inammatory bowel disease by scavenging ROS and down-regulating proinfammatory cytokines. 34Naha et al. synthesized dextran-coated CeO 2 nanoparticles (d = 4.8 nm) and applied them to CT diagnosis of gastrointestinal tract and inammatory bowel disease. 35The X-ray attenuation was ∼1.2 times greater than that of the commercial iodine contrast agent Iopamidol.Jia et al. synthesized doxorubicin-loaded upconversion core@mesoporous CeO x shell nanoplatforms (d = ∼48 nm) for tumor diagnosis via CT and the synergistic chemophotodynamic therapy of tumor. 36Feng et al. synthesized citric acid-coated CeO 2 nanoparticles (d = ∼3 nm) as a renoprotective contrast agent and successfully applied them to in vivo spectral CT angiography. 37Youn et al. synthesized CeO 2 nanoparticles (d = 3.5 nm) and nanorods (9.4 × 130 nm), and compared their therapeutic effects.Compared to the nanoparticles, the nanorods demonstrated better effects on reducing cerebral edema. 38erein, ultrasmall CeO 2 nanoparticles (d = 1-3 nm) coated with hydrophilic and biocompatible polymers, namely, poly(acrylic acid) (PAA) and poly(acrylic acid-co-maleic acid) (PAAMA), were synthesized using the one-pot polyol method.Their particle diameters were less than those [32][33][34][35][36][37][38][39] of the previously investigated nanoparticles.Notably, smaller CeO 2 nanoparticles in particle size can exhibit a higher X-ray attenuation efficiency due to their more effective X-ray attenuation and more powerful antioxidant effect because of their higher amounts of Ce 4+ on nanoparticle surfaces.Therefore, ultrasmall CeO 2 nanoparticles synthesized herein can act as highly sensitive radioprotective or theranostic X-ray contrast agents.The polymer-coated ultrasmall CeO 2 nanoparticles were characterized using various techniques.Cellular cytotoxicity was assessed to conrm their suitability for biomedical applications.The Xray attenuation properties were characterized by measuring phantom images.The CT images in vivo were measured before and aer intravenous (IV) and intraperitoneal (IP) injections to conrm the potential of the CeO 2 nanoparticles as X-ray contrast agents.Finally, their antioxidant effect was evaluated by measuring the removal of hydrogen peroxide (H 2 O 2 ) in the oxidation reaction of rhodamine B (Rh B) under H 2 O 2 /365 nm ultraviolet (UV) irradiation with and without the nanoparticles.

Results and discussion
Colloidal stability, particle diameter, hydrodynamic diameter, zeta potential, and crystallinity The PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles, exhibiting colloidal stability, were successfully prepared using a simple one-pot polyol method (Fig. S1 †), as conrmed by the below-described characterization methods.
Transparent nanoparticles were suspended in aqueous media, which did not undergo precipitation aer synthesis (>1.5 years), indicating excellent colloidal stability (Fig. 1a).The high negative average zeta potentials (z avg ) of −48.3 and −43.0 mV for the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles in aqueous media, respectively (Fig. 1b and Table 1), conrmed their excellent colloidal stability in aqueous media.The colloidal dispersion was also conrmed by Tyndall effect (Fig. S2 †); light scattering was observed only for nanoparticle suspension samples owing to the collision between the nanoparticle colloids and laser light, whereas light scattering was not observed in triple-distilled water.
High-resolution transmission electron microscopy (HRTEM) images of polymer-coated CeO 2 nanoparticles revealed nearly monodisperse particle diameter distributions (Fig. 2a(i), a(ii), b(i) and b(ii)) in which (i) and (ii) label PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles, respectively.Additional HRTEM images are provided in ESI (Fig. S3 and S4 †).The nanoparticle dispersions were conrmed by elemental mapping in the highangle annular dark eld-scanning transmission electron microscope (HAADF-STEM) mode (Fig. 2c(i) and (ii)), which revealed the uniform elemental distribution of Ce (Fig. 2d(i) and (ii)) in HAADF-STEM images.X-ray energy dispersive spectroscopy spectra (Fig. S5a and b †) conrmed the presence of Ce in the nanoparticles.The average particle diameters (d avg ) for PAAand PAAMA-coated ultrasmall CeO 2 nanoparticles were  Paper RSC Advances estimated to be 1.8 and 2.0 nm, respectively, based on the lognormal function ts to the observed particle diameter distributions (Fig. 2e and Table 1).The average hydrodynamic diameter (a avg ) values of the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles were estimated to be 14.5 and 15.5 nm, respectively, based on the log-normal function ts to the observed dynamic light scattering (DLS) patterns (Fig. 2f).The large hydrodynamic diameter of the nanoparticles was attributed to the PAA and PAAMA coatings on the nanoparticle surfaces and accompanying hydration of a large amount of water.Each monomer in PAA comprises one carboxyl group.PAAMA comprises almost equal numbers of acrylic acid (AA) and maleic acid (MA) monomers, and each of the AA and MA monomers comprises one and two carboxyl groups, respectively.These numerous carboxyl groups possibly lead to strong binding between the polymers and nanoparticles via electrostatic (i.e., hard acid-base) interaction, consequently supporting their observed excellent colloidal stability in aqueous media.

Fourier transform-infrared (FT-IR) absorption spectra and TGA curves
The surface coating of PAA and PAAMA on the nanoparticle surfaces was conrmed by FT-IR absorption spectra (Fig. 4a and  b, respectively).The surface-coating amount was obtained from the TGA curves (Fig. 4c).As shown in Fig. 4a and b, C-H symmetric stretching vibration at ∼2930 cm −1 , COO − antisymmetric stretching vibration at ∼1550 cm −1 , and COO − symmetric stretching vibration at ∼1395 cm −1 conrmed the successful coating of PAA and PAAMA on the CeO 2 nanoparticle surfaces.The red-shis and splittings 43 of the C]O symmetric stretching vibrations of the -COOH groups of free PAA and PAAMA at ∼1695 cm −1 into the symmetric and antisymmetric COO − stretching vibrations in the FT-IR absorption spectra of the nanoparticle samples conrmed electrostatic (i.e., hard acid-base) bonding 44 between the COO − groups of PAA and PAAMA and Ce 4+ on the nanoparticle surfaces, as observed in other metallic oxide nanoparticles. 45,46Table S1 † also summarizes the observed FT-IR absorption frequencies.The red-shis of the COO − antisymmetric and symmetric stretching vibrations from the C]O vibrations were ∼140 and ∼300 cm −1 (Table S1 †), respectively, conrming the strong bonding.In addition, because PAA and PAAMA comprise many -COOH groups, they can bind to a nanoparticle via multiple bonds, as schematically drawn in Fig. 4d, consequently leading to the strong bonding of the polymer to the CeO 2 nanoparticles and the long-term colloidal stability of the polymer-coated nanoparticles in aqueous media (i.e., no precipitation aer synthesis, >1.5 years).
The observed good colloidal stability conrmed that a sufficient amount of polymers should be coated on the CeO 2 nanoparticle surfaces, which was conrmed from the TGA curves in Fig. 4c.The surface-coating amount (S) was estimated in wt% by measuring the mass losses aer heating from ∼100 °C up to 900 °C because the initial mass drops (i.e., 6% and 11%) up to ∼100 °C were attributed to the desorption of water and air.Graing density (s), 47,48 dened as the average number of polymers coating a unit surface area of a nanoparticle, was obtained using the bulk density of CeO 2 (7.132 g cm −3 ), 49 d avg values estimated from HRTEM images, and aforementioned S values.The average number (N polymer ) of polymers coating a nanoparticle was determined as the product of s and nanoparticle surface area (=pd avg 2 ).Table 1 summarizes the surface-coating results.

In vitro cytotoxicity results
The PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles exhibited very low in vitro cellular cytotoxicity (Fig. 5a and b), thereby demonstrating their suitability for biomedical applications.The high cell viability (>90%) of human prostate cancer (DU145) and normal mouse hepatocyte (NCTC1469) cells up to 500 mM [Ce] 48 h aer incubation with nanoparticle samples was observed.Cell morphologies were examined using an optical microscope (Fig. 5c and d).The cell morphologies of the treated cells were similar to those of the control cells, which was consistent with the observed very low cellular cytotoxicity of the nanoparticles.

Antioxidant effect
To evaluate the antioxidant effect of the PAA-and PAAMAcoated ultrasmall CeO ).The solution photographs (Fig. 6) and PL spectra (Fig. 7) were measured at intervals of 6 h up to 24 h.1][52][53][54] However, Rh B undergoes rapid decomposition in the presence of the oxidizing agent H 2 O 2 under UV irradiation according to the following oxidation reaction, 55 A similar oxidation reaction of Rh B was observed in the Rh B/H 2 O 2 /hydroxylamine (HA) system in which HA reacted with H 2 O 2 to generate hydroxyl radical (cOH) to decompose Rh B. 55 As shown in Fig. 6, solution-a exhibited an unnoticeable degradation of pink color up to 24 h, indicating that Rh B negligibly decomposed without H 2 O 2 regardless of 365 nm UV irradiation (power = 15 W).Solutions-f and -g also exhibited unnoticeable pink color degradation up to 24 h, indicating that Rh B did not undergo decomposition by the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles regardless of the UV irradiation.Solutions-b, -c, and -d were transparent (i.e., no color) because of the absence of Rh B in solutions, indicating that the pink color was solely attributed to Rh B, and not H 2 O 2 and PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles.In  Therefore, the net antioxidant reaction was as follows: The antioxidant effects of the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles were quantitatively investigated by recording PL spectra (Fig. 7a-f).Solutions-a, -f, and -g exhibited an unnoticeable PL intensity drop with time up to 24 h (Fig. 7a, c and d, respectively), which was consistent with the observation of unnoticeable pink color degradation in the solution photographs in Fig. 6a, f and g, respectively.The PL spectra of solutions-b, -c, and -d were not measured because Rh B was absent in the solutions.Solution-e exhibited a rapid drop in the PL intensity with time (Fig. 7b), whereas solutions-h and -i containing nanoparticles exhibited a delayed drop in the PL intensity (Fig. 7e and f, respectively), conrming the antioxidant effect of the nanoparticles.To quantitatively evaluate the degradation efficiency (%) of Rh B with time, dened as 100 (I 0 − I t )/I 0 , where I t is the PL intensity at time t, it was plotted as a function of time in Fig. 7g.Solutions-a, -f, and -g exhibited a negligible degradation efficiency of Rh B overtime.Solution-e rapidly exhibited ∼100% degradation efficiency of Rh B at 12 h, whereas solutions-h and -i exhibited only ∼78% degradation efficiency of Rh B at 24 h due to the antioxidant effect of the nanoparticles.This result conrmed the antioxidant effect of the PAA-and PAAMA-coated CeO 2 nanoparticles; therefore, these nanoparticles exhibited potential as radioprotective or theranostic X-ray contrast agents by removing ROS (i.e., H 2 O 2 and cOH) produced by X-rays during X-ray scan.

X-ray attenuation: phantom images
The contrasts of the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles in the X-ray phantom images were brighter than those of a commercial molecular iodine(I) contrast agent Ultravist at similar atomic concentrations of [Ce] and [I] (Fig. 8a), demonstrating that the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles were superior than Ultravist.This result was attributed to the higher linear X-ray attenuation coefficient of Ce than that of I (Fig. 8b). 22To quantitatively discuss this result, X-ray attenuation estimated from X-ray phantom images was plotted as a function of the atomic concentration.The X-ray attenuation of the PAA-and PAAMAcoated ultrasmall CeO 2 nanoparticles was greater than that of Ultravist at the same atomic concentration of [Ce] and [I] at 70 kV p (Fig. 8c).In addition, Fig. 8d shows the X-ray attenuation of the nanoparticles as a function of the number density: the X-ray attenuation at the same number density was greater than that observed at the same atomic concentration: therefore, nanoparticle contrast agents can provide considerably higher contrast enhancement than molecular agents at the same number density, making the nanoparticle contrast agents superior than molecular contrast agents.The number density was estimated by multiplying the molar atomic concentration with 6.02 × 10 23 /N atom , where N atom is the number of X-ray attenuating atoms per molecule or nanoparticle; N atom is three for Ultravist, and ∼(1/3) (d avg /h) 3  As a key parameter for comparing materials as X-ray contrast agents, the X-ray attenuation efficiency (h), dened as the X-ray attenuation per molar concentration [Hounseld units (HU)/ mM] or per number density [HU/(1/L)], was estimated from the slopes in Fig. 8c and d, respectively.Table 2 summarizes the results.The h values of the nanoparticles were 1.3 and 68 times greater than those of Ultravist in terms of the molar atomic concentration and number density, respectively.In addition, the h value estimated herein was greater than those 35,39 of larger CeO 2 nanoparticles (Fig. 8e).This result was attributed to the particle size effect, i.e., smaller nanoparticles can attenuate Xrays more effectively than larger nanoparticles because of the exponential decay of X-rays along the penetration depth.Therefore, the results obtained herein revealed that the PAAand PAAMA-coated ultrasmall CeO 2 nanoparticles demonstrate promise as highly sensitive X-ray contrast agents.

In vivo CT images
The potential of the nanoparticles as X-ray contrast agents was further conrmed in vivo using the PAA-coated ultrasmall CeO 2 nanoparticles.The nanoparticles dispersed in aqueous media were injected via two routes: intravenously (IV) via the mice tails and intraperitoneally (IP).The CT images were recorded before and aer injection using an injection dose of ∼0.1 mmol Ce per kg, which was less than that (>1 mmol I per kg) 6,20 of the iodine contrast agents.Positive contrast enhancement was observed in the mice bladder aer IV and IP injections even at an injection dose of ∼10 times less than those of iodine contrast agents (Fig. 9a).The contrasts were quantitatively shown in Fig. 9b by plotting the signal-to-noise ratio (SNR) of a region of interest (ROI) at the bladder as a function of time.Compared with the IP injection, the IV injection exhibited a more rapid SNR increase and drop due to the faster excretion of the nanoparticles aer the IV injection than that aer the IP injection. 60,61This in vivo result conrmed that the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles demonstrate potential as CT contrast agents.

Experimental
Synthesis of polymer-coated ultrasmall CeO 2 nanoparticles (polymer = PAA and PAAMA) The schematic of the one-pot polyol synthesis 58,62 is shown in Fig. S1, † and details of the synthesis are provided in ESI.† In this method, triethylene glycol (TEG) as solvent suppressed the particle size growth, leading to TEG-coated ultrasmall CeO 2 nanoparticles.Then, TEG was replaced with PAA (or PAAMA) because -COOH groups of the PAA (or PAAMA) can more strongly bind to the CeO 2 nanoparticles than -OH group of the TEG.

General characterization
The synthesized nanoparticles were characterized as described in detail in previous studies. 58,62The Ce

In vitro cell viability measurements
The in vitro cytotoxicity of polymer-coated ultrasmall CeO 2 nanoparticles was measured using the DU145 and NCTC1469 X-ray phantom image measurements X-ray attenuation was estimated by measuring X-ray phantom images using a micro-CT scanner (Inveon, Siemens Healthcare, Erlangen, Germany) at an X-ray source voltage of 70 kV p , an Xray source current of 280 mA, and an imaging time per frame of 300 ms.It was estimated in HU with respect to that of water with 0.0 HU using the formula HU = 1000 (m sample − m water )/

In vivo CT image measurements
Female ICR mice (ICR = Institute of Cancer Research, USA) with a weight of ∼40 g were injected with 0.1 mmol Ce per kg and used for imaging.For imaging, the mice were anesthetized using 1.5% isourane in oxygen, and measurements were conducted before and aer IV injection with the PAA-coated ultrasmall CeO 2 nanoparticles dispersed in aqueous media into the mice tails under the following conditions: number of mice (N) = 2, X-ray source voltage = 70 kV p , X-ray source current = 280 mA, imaging time per frame = 1700 ms, thickness = 0.148 nm, and resolution = 512 × 512.The measurements were also conducted before and aer IP injection (200 mL).Aer measurements, the mice were revived from anesthesia and placed in a cage with free access to food and water.

Conclusions
Hydrophilic and biocompatible PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles (d avg values of 1.8 and 2.0 nm, respectively, the smallest size reported thus far) were synthesized using the one-pot polyol method.
(2) Their X-ray attenuation efficiency was 1.3 times greater than that of Ultravist.Furthermore, it was greater than those of various large CeO 2 nanoparticles reported previously.
(3) They exhibited an antioxidant effect for the removal of H 2 O 2 .
(4) The results from in in vivo mice experiments conrmed that the nanoparticles exhibited contrast enhancement aer IV and IP injections.All these results suggested that PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles are highly sensitive X-ray contrast agents with antioxidant effects.methodology.Ji-ung Yang and Ji Ae Park: IP injection CT image acquisition.Byeong Woo Yang: validation.Kwon Seok Chae: cell viability assay.Sung-Wook Nam: funding.Yongmin Chang and Gang Ho Lee: funding, supervision and writing.

Fig. 1
Fig. 1 (a) Photographs of PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles dispersed in aqueous media and water.(b) Zeta potential (z) curves and Gaussian function fits to obtain z avg .

Fig. 3
Fig. 3 XRD patterns of the powder samples of the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles (a) before and (b) after TGA up to 900 °C under airflow.The peaks at the top of the peaks are (hkl) Miller indices of bulk CeO 2 with an FCC crystal structure.

Fig. 4
Fig. 4 FT-IR absorption spectra of (a) free PAA and PAA-coated ultrasmall CeO 2 nanoparticles and (b) free PAAMA and PAAMA-coated ultrasmall CeO 2 nanoparticles."as" and "ss" indicate the antisymmetric and symmetric stretching vibrations of COO − , respectively.(c) TGA curves of the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles under air flow.(d) Schematic of the coating structures of PAA and PAAMA polymers on the nanoparticle surfaces via electrostatic (i.e., hard acid-base) bonding between the COO − groups of the polymers and Ce 4+ on the nanoparticle surfaces (the minor Ce 3+ ions also exist on the nanoparticle surfaces, but only the major Ce 4+ ions were displayed on the nanoparticle surfaces).

Fig. 5
Fig. 5 In vitro cell viability of (a) NCTC1469 and (b) DU145 cells and optical microscopy images of (c) NCTC1469 and (d) DU145 cells 48 h after incubation with the PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles up to 500 mM [Ce].Scale bar = 70 nm.

Fig. 8
Fig. 8 (a) X-ray phantom images of Ultravist and PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles dispersed in aqueous media at an X-ray source voltage of 70 kV p .(b) Plot of the linear attenuation coefficients of Ce and I versus radiation photon energy.Plots of the X-ray attenuation as a function of the (c) atomic concentrations of [Ce] and [I] and (d) number density of the nanoparticles and Ultravist: slopes of the dotted lines correspond to X-ray attenuation efficiencies (h).(e) Comparison of h values: dextran-coated CeO 2 nanoparticles (d = 4.8 nm, 80 kV p ), 35 porous Ce 2 (CO 3 ) 2 O$H 2 O nanoparticles (d = 196.6 nm, 80 kV p ), 39 and polymer-coated ultrasmall CeO 2 nanoparticles [d = (1.8+ 2.0)/2 = 1.9 nm, 70 kV p ] (this study).Water: 0 HU.

Table 1
Physicochemical properties of PAA-and PAAMA-coated ultrasmall CeO 2 nanoparticles a Average coating amount of polymers per nanoparticle in wt%.b Graing density, i.e., average number of polymers coating a nanoparticle unit surface area.c Average number of polymers coating a nanoparticle.